Coumarins as Modulators of the Keap1/Nrf2/ARE Signaling Pathway

The Keap1/Nrf2/ARE system is a central defensive mechanism against oxidative stress which plays a key role in the pathogenesis and progression of many diseases. Nrf2 is a redox-sensitive transcription factor controlling a variety of downstream antioxidant and cytodefensive genes. Nrf2 has a powerful anti-inflammatory activity mediated via modulating NF-κB. Therefore, pharmacological activation of Nrf2 is a promising therapeutic strategy for the treatment/prevention of several diseases that are underlined by both oxidative stress and inflammation. Coumarins are natural products with promising pharmacological activities, including antioxidant, anticancer, antimicrobial, and anti-inflammatory efficacies. Coumarins are found in many plants, fungi, and bacteria and have been widely used as complementary and alternative medicines. Some coumarins have shown an ability to activate Nrf2 signaling in different cells and animal models. The present review compiles the research findings of seventeen coumarin derivatives of plant origin (imperatorin, visnagin, urolithin B, urolithin A, scopoletin, esculin, esculetin, umbelliferone, fraxetin, fraxin, daphnetin, anomalin, wedelolactone, glycycoumarin, osthole, hydrangenol, and isoimperatorin) as antioxidant and anti-inflammatory agents, emphasizing the role of Nrf2 activation in their pharmacological activities. Additionally, molecular docking simulations were utilized to investigate the potential binding mode of these coumarins with Keap1 as a strategy to disrupt Keap1/Nrf2 protein-protein interaction and activate Nrf2 signaling.


Introduction
Coumarins are organic compounds in the benzopyrone class. This group comprises a large number of compounds that are widely distributed in the plant kingdom and have the highest concentration in fruits, seeds, roots, and leaves [1]. The name coumarin has been derived from the French word "Coumarou," the common name of tonka beans (Dipteryx odorata) [2, 3]. The number of coumarins identified as secondary metabolites in bacteria, fungi, and about 150 species of plants has been estimated to be more than 1300 compounds [4]. Coumarins are most abundant in those plants taxonomically assigned to the Apiaceae, Asteraceae, and Rutaceae families and play a significant role in human health. Coumarins have been widely used in complementary and alternative medicine and possess a diversity of pharmacological activities with low cost and few side effects [5][6][7]. Carrot, cherries, citrus fruits, apricots, celery, parsnip, and strawberries, as well as spices like cinnamon and fennels are some of the commonly consumed coumarincontaining phytofoods [8].
Natural coumarins are classified into 6 main types based on their chemical structure. These include simple coumarins, furanocoumarins, dihydrofuranocoumarins, phenylcoumarins, pyranocoumarins, and biscoumarins [6]. All have a coumarin core and are characterized by structural diversity which could be considered for drug discovery and development of therapeutic agents for multiple diseases [9][10][11][12]. In plants, coumarins have been suggested to function as growth regulators and bacterio-and fungistatic agents [13]. In addition, coumarins possess a broad range of pharmacological activities basically relying on the type of coumarin nucleus.
The antioxidant and anti-inflammatory activities of coumarins have been well-acknowledged in several in vitro and in vivo studies [36,37]. Coumarins suppress oxidative stress through their ability to scavenge reactive oxygen species (ROS) and inhibit neutrophil-dependent superoxide anion generation and lipid peroxidation. Moreover, coumarins can effectively reduce tissue edema-associated inflammation through suppressing both lipoxygenase and cyclooxygenase enzymatic activities and prostaglandin synthesis and release [20,[38][39][40].
Oxidative stress is a state of imbalance between the production of free radicals and their degradation by antioxidants. This redox imbalance occurs as a result of increased ROS generation and diminished antioxidant defenses. Although produced normally through different metabolic processes, excess ROS can provoke inflammation and damage lipids, proteins, and other cellular macromolecules, leading to oxidative stress and cell death. Therefore, oxidative stress is implicated in the pathogenesis of a wide range of metabolic disorders and chronic diseases [41][42][43]. Given their ability to suppress excessive ROS generation and enhance antioxidants [30,34,44], the pharmacologic effects of coumarins could be mediated through their antioxidant efficacy. The present review presents an overview of the modulatory role of a number of plant-derived coumarins (Figure 1) on nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor which protects against oxidative injury and inflammation [45]. In addition, we investigated the potential binding mode of coumarins to Kelch-like ECH-associated protein 1 (Keap1) as a strategy to disrupt Keap1/Nrf2 protein-protein interaction (PPI) using molecular docking simulations.

Keap1/Nrf2/ARE Signaling Pathway
Nrf2 is a transcription factor generally known to enhance the cellular defense system to counteract oxidative injury and inflammation. In conditions without oxidative stimuli, Nrf2 exists in the cytoplasm sequestered by Keap1 [46]. Keap1 mediates Nrf2 ubiquitination and subsequent proteasomal degradation through acting as an adaptor molecule for the CUL-E3 ligase. The dissociation of Keap1 from the CUL-E3 ligase is elicited upon exposure to electrophilic/oxidative stress which modifies the cysteine residues of Keap1, in particular Cys151, leading to accumulation of Nrf2 [47]. As a result, Nrf2 liberates and translocates into the nucleus where it binds to the antioxidant response element (ARE) and promotes the transcription of antioxidant genes [48] (Figure 2). Nrf2-target antioxidant genes include heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO1), γglutamyl cysteine ligase modulatory and catalytic subunits (GCLM and GCLC, respectively), and ferritin, which function to maintain the oxidant/antioxidant balance inside the cells [48]. Thus, activation of Nrf2 is an effective strategy to suppress oxidative stress.
Besides attenuation of oxidative stress, Nrf2 activation can effectively prevent inflammation. Nrf2 signaling is regarded as the most sensitive redox pathway linked to oxidative injury and nuclear factor-κB (NF-κB), a master regulator of proinflammatory mediators. Both are redox-sensitive factors where NF-κB is activated in oxidative stress conditions, and the lack of Nrf2 resulted in increased oxidative/nitrosative stress and subsequently amplification of cytokine production [50]. The crosstalk between Nrf2 and NF-κB has been reviewed by Wardyn et al. [51]. The lack of Nrf2 can aggravate NF-κB activity leading to increased inflammatory cytokine release [52], whereas Nrf2 upregulation resulted in diminished inflammatory responses in rodent models of liver and kidney injury [53][54][55][56][57][58][59][60]. The Nrf2 target gene HO-1 has been demonstrated to inhibit NF-κB-mediated transcription of adhesion molecules possibly through decreasing free intracellular iron in endothelial cells [61]. NF-κB activity can regulate Nrf2-mediated antioxidant gene expression. In this context, Yu et al. [62] have demonstrated that overexpression of the canonical NF-κB subunit p65 increased nuclear Keap1 levels resulting in decreased Nrf2/ARE signaling. Given that Keap1 is mostly localized in the cytosol and proteins larger than 40 kDa cannot enter through the nuclear envelope, the nuclear translocation of Keap1 has been suggested to occur via interaction with karyopherin alpha 6 (KPNA6). In this context, overexpression of KPNA6 has been associated with decreased HO-1 and NQO1 [63]. Although different mechanisms of the inhibitory effect of NF-κB p65 on Nrf2 have been postulated, a competition for the transcriptional coactivator CBP-p300 complex is the best supported mechanism. CBP-p300 has an intrinsic acetyl transferase activity and acetylates Nrf2 and p65 [64,65].

The Modulatory Effect of Coumarins on Nrf2
In this section, we reviewed studies showing the role of Nrf2 signaling in mediating the pharmacologic effects of coumarins. Only coumarin derivatives reported to modulate Nrf2 signaling were included.

Oxidative Medicine and Cellular Longevity
In ovalbumin-(OA-) challenged mice and LPS-induced dendritic cells (DCs), the antiallergic and anti-inflammatory effects of IMP were demonstrated [72]. In asthmatic mice, IMP, in a dose-dependent manner, reduced IgE levels, airway hyperresponsiveness, and Th2 cytokines and increased IL-10-producing T cells. In LPS-stimulated DCs, IMP increased IL-10 and suppressed the release of proinflammatory cytokines [72]. Li et al. have reported that IMP effectively diminished COX-2, IL-6, TNF-α, and iNOS expression via downregulation of NF-κB and JAK/STAT signaling in alveolar macrophages [74]. In addition, IMP relieved zymosaninduced immune cell infiltration in mice, thereby attenuating lung inflammation, edema, and fibrosis [74]. Although these studies have documented the potent anti-inflammatory activity of IMP, none of them have pointed to the involvement of Nrf2 signaling. The ability of IMP to activate Nrf2 signaling has been evaluated in few studies. By using HepG2 stably transfected with ARE reporter and Nrf2 knockout mice, Prince et al. have studied whether IMP activates hepatic Nrf2. The results showed that IMP increased hepatic GST activity via the Nrf2/ARE mechanism [75]. Hu et al. reported that IMP-induced Nrf2 activation effectively upregulated its downstream antioxidants, possibly offering cellular protection to the heart from injury induced by arsenic trioxide (As 2 O 3 ) [76]. Very recently, Xian et al. investigated the protective effect of IMP against excess ROS and chronic airway inflammation in OA-administered mice [77]. Treatment with IMP suppressed ROS, proinflammatory cytokines, inflammatory cell infiltration, collagen deposition, and goblet cell hyperplasia. These effects have been suggested to be mediated via modulation of Nrf2 signaling following IMP administration [77].
3.2. Visnagin. Visnagin (4-methoxy-7-methylfuro[3,2-g] chromen-5-one) is a bioactive compound extracted from Ammi visnaga fruits [78]. Visnagin possesses widespread pharmacological activities, including hypotensive and smooth muscle relaxation. In a dose-dependent manner, visnagin decreased blood pressure when administered intravenously. In isolated mesenteric arteries precontracted with noradrenaline, visnagin treatment resulted in a concentrationdependent relaxation [79]. In isolated rat aortic rings, visnagin inhibited the vascular smooth muscle contraction induced by different agents [80]. Visnagin has also shown protective effects against doxorubicin cardiotoxicity mediated via cytochrome P450 family 1 (CYP1) inhibition [81] and modulation of mitochondrial malate dehydrogenase [82]. In addition, visnagin prevented the deposition of renal crystals in hyperoxaluric rats [83]. Besides these activities, the antioxidant and anti-inflammatory activities of visnagin have been demonstrated. In this context, Lee   Oxidative Medicine and Cellular Longevity α, IL-1β, iNOS, IFN-γ, and IL-6 expression via a NF-κBdependent mechanism and increases the production of the anti-inflammatory cytokine IL-10 [84]. A recent study by Khalil et al. evaluated the anti-inflammatory activity of visnagin using molecular docking simulation and in vivo and in vitro assays [85]. Visnagin exhibited a strong antiinflammatory activity with a high safety profile in vivo and showed an in vitro selective COX-2 inhibition [85]. The role of Nrf2 in mediating the anti-inflammatory and antioxidant activities of visnagin has been recently reported by Pasari et al. using a model of cerulein-induced acute pancreatitis in mice [86]. Visnagin decreased the expression of IL-1β, TNF-α, IL-6, and IL-17 in a dose-dependent manner. Importantly, visnagin enhanced the antioxidant defenses by effective upregulation of Nrf2 and mitigated pancreatic inflammation through suppression of the NF-κB expression in the acinar cells. Additionally, visnagin inhibited the release of inflammatory cytokines in pulmonary and intestinal tissues [86].
3.3. Urolithin B. Urolithin B (3-hydroxybenzo[c]chromen-6one) is one of the gut microbial metabolites of ellagitannins, a class of compounds found in many plants, including medicinal herbs, pomegranates, and tropical fruits [87,88]. Ellagitannin is hydrolyzed to ellagic acid which is then catabolized by microflora in the intestinal tract into urolithin derivatives (urolithin A-D) [89]. Urolithin B is the final product catabolized among the urolithin derivatives that possesses anticancer activity on prostate [90] and colon cancer [91]. Treatment with urolithin B induced apoptosis of HT-29 colon cancer cells in vitro [91]. The anticancer activity of urolithin B was mediated through disruption of the mitochondrial membrane potential and activation of caspases 8, 9, and 3 [91]. In addition, urolithin B exhibited potent anti-inflammatory [92] and antioxidant effects [93] and has been reported to attenuate neurotoxicity in Alzheimer's disease by preventing β-amyloid fibrillation in vitro [94]. The dual antiinflammatory and antioxidant effect of urolithin B has been recently investigated by Lee et al. [92] in microglia challenged with LPS. Urolithin B reduced cytokine release and NO production, while it significantly increased the expression of IL-10. In addition, urolithin B significantly decreased TNF-α, IL-6, and NO production in stimulated BV2 cells. Additionally, the in vivo studies reported that urolithin B inhibited LPS-induced microglia activation in the mouse brain [92]. The study has also scrutinized the antioxidant activity of urolithin B. The results showed a strong antioxidant activity mediated by reducing intracellular ROS production and NADPH oxidase subunit expression through upregulation of Nrf2/ARE signaling and HO-1 expression. Interestingly, urolithin B suppressed the activity of NF-κB by reducing IκBα phosphorylation. In addition, urolithin B inhibited both ERK and JNK phosphorylation while it enhanced the AMPK phosphorylation which is associated with a dual antiinflammatory and antioxidant activity [92].

Urolithin A. Urolithin A (3,8-dihydroxybenzo[c]
chromen-6-one) is the one of the main microbiota metabolites of pomegranate ellagitannins. It is characterized by high safety as evidenced by a previous study carried out by Heilman et al. [95]. Urolithin A has a wide range of bioactivities, such as antioxidant [96], anti-inflammatory [97,98], and anticancer effects [99]. In human colonic fibroblasts stimulated with IL-1β, urolithin A inhibited PGE2 production, downregulated COX-2, and suppressed NF-κB nuclear translocation [97]. In human aortic endothelial cells exposed to TNF-α, urolithin A showed a great ability to inhibit monocyte adhesion, chemokine expression, and cell migration [98]. Urolithin A improved the gut microbiota in high fat diet-(HFD-) fed rats and reversed the LPS-induced inflammatory response in Caco2 cells [100]. Fu et al. have conducted a study to examine the role of urolithin A on IL-1β-induced inflammatory response in human osteoarthritis through in vitro and in vivo approaches. In human chondrocytes in vitro, urolithin A prevented the IL-1β-induced overproduction of TNF-α, IL-6, PGE2, COX-2, iNOS, and NO in a concentration-dependent manner via suppressing NF-κB activation [101]. Another recent study carried out by Gong et al. concluded that urolithin A attenuates memory impairment and neuroinflammation in APP/PS1 mice via suppressing p38 MAPK and NF-κB p65 activation along with enhancing cerebral AMPK activation [102]. Two recent studies have reported the impact of Nrf2 activation by urolithin A. The first study was done by Liu et al. who demonstrated that urolithin A is a promising antiaging agent through potent inhibition of intracellular ROS promoted by activation of the Nrf2-mediated antioxidative response [103]. The second study done by Singh et al. concluded that urolithin A can be used for the treatment of colitis through remedying barrier dysfunction [104]. This study showed the antiinflammatory effects of urolithin A and highlighted the role of the activation of Nrf2-dependent pathways [104].
3.5. Scopoletin. Scopoletin (7-hydroxy-6-methoxychromen-2-one) is a natural coumarin found in many medicinal plants, including members of the genus Scopolia as well as species of the Artemisia, Brunfelsia, Solanum, and Mallotus and other genera. Scopoletin is present in many edible plants and foods, such as oats, garlic, lemon, grapefruit, celery, red pepper, chili pepper, carrots, chicory, and bael [105][106][107]. It possesses many pharmacological functions, including antimicrobial [108], antiaging [109], anti-inflammatory [110,111], and antioxidant effects [112,113]. Scopoletin is also known for its cytotoxic activity against different cancer cells [114,115]. Scopoletin has also shown antihyperglycemic activity in diabetic mice and inhibited α-glucosidase in vitro [116]. The antidiabetic activity of scopoletin has been suggested to be exerted via inhibition of the carbohydrate digestive enzymes [116]. In the same context, scopoletin improved insulin sensitivity and enhanced glucose uptake through activation of GLUT4 translocation and PI3K and AMPK signaling in 3T3-L1 adipocytes in vitro [117]. The supplementation of scopoletin prevented hepatic steatosis in diabetic mice by inhibiting lipid biosynthesis and TLR4-MyD88 pathways [118]. The anti-inflammatory property of scopoletin was shown in vivo in various animal studies [110,111,118,119]. The anti-inflammatory activity of scopoletin has been reported to involve suppression of myeloperoxidase (MPO), a neutrophil infiltration biomarker; adenosine-deaminase (ADA); TNF-α; IL-1β; and NO via inhibition of the NF-κB and p38 MAPK phosphorylation. In a rat model of osteoarthritis, scopoletin downregulated collagenases and reduced the proinflammatory mediators in a dose-dependent manner [119]. In human fibroblasts, scopoletin inhibited p38 phosphorylation, MMP-1, NF-κB, MAPK, and the mRNA abundance of IL-1α and TNF-α [120]. In a mouse model of cerulein-induced acute pancreatitis and lung injury, scopoletin suppressed pancreatic and pulmonary TNF-α, IL-1β, mast cell activation, and NF-κB signaling [111]. The ability of scopoletin to upregulate Nrf2/HO-1 signaling has been supported by few studies. Given the antidiabetic efficacy of scopoletin, Chang et al. have investigated its insulin sensitizing and antiglycation effects in diabetic rats, pointing to the role of Nrf2 signaling [121]. Scopoletin suppressed the formation of advanced glycation end products (AGEs), hyperglycemia, and insulin resistance and enhanced Nrf2, Akt, and GLUT2 in hepatocytes [121]. Very recently, Narasimhan et al. reported that scopoletin protected against oxidative stress and apoptosis induced by rotenone via Nrf2 activation and investigated its neuroprotective effects for Parkinson's disease in a rat model and in vitro using SH-SY5Y cells [122].
Zhang et al. have demonstrated the protective effect of daphnetin against cisplatin nephrotoxicity via suppressing oxidative injury and inflammation [132]. Daphnetin significantly inhibited cisplatin-induced ROS generation, lipid peroxidation, NF-κB activation, and the levels of IL-1β and TNF-α production in a dose-dependent manner. These effects have been accompanied with upregulation of Nrf2 and HO-1 expression [132]. Another study by Liu et al. reported that daphnetin protected against NAFLD in vitro in oleic acid-treated hepatocytes through Nrf2 activation while it effectively decreased CYP2E1 and CYP4A expression [133]. Daphnetin showed a potent inhibitory effect on oleic acid-induced ROS generation and promoted glucose uptake, insulin sensitivity, and PI3K/Akt signaling in hepatocytes [133]. In support of these findings, daphnetin mitigated oxidative stress in human lung epithelial cells exposed to arsenic through Keap1 protein downregulation and marked activation of the Nrf2-dependent antioxidant response with a dramatic upregulation of the ARE in a dose-dependent manner [134]. Daphnetin inhibited oxidative stress and inflammatory response in high glucose-(HG-) stimulated human glomerular mesangial cells (MCs) [135]. Daphnetin strikingly reduced ROS and decreased the production of IL-1β, IL-6, and TNF-α via suppression of the NF-κB pathway. Mechanistically, daphnetin positively upregulated Nrf2 while it inhibited the expression Keap1 in HG-stimulated MCs [135]. Zhi et al. reported that daphnetin protected hippocampal neurons exposed to oxygen-glucose deprivation-induced injury against I/R via marked enhancement of the nuclear translocation of Nrf2 and HO-1 expression [136]. Moreover, Nrf2 knockdown blocked the protective effect of daphnetin on I/R in hippocampal neurons, confirming the critical role of Nrf2/HO-1 signaling activation in the neuroprotective effect of daphnetin [136]. Lv and coworkers demonstrated that daphnetin effectively inhibited cytochrome c release and NLRP3 inflammasome activation through upregulation of the Nrf2 nuclear translocation along with Keap1 protein downregulation [137]. Additionally, daphnetin suppressed ROS generation induced by tert-butyl hydroperoxide (t-BHP) which is mostly blocked in Nrf2 knockout macrophages. Accordingly, daphnetin has a protective role against t-BHP-induced oxidative injury via the Nrf2/ARE signaling pathway [137]. Furthermore, daphnetin ameliorated carbon tetrachloride-(CCl 4 -) induced hepatotoxicity in rats through induction of the nuclear translocation of Nrf2, thus inducing HO-1 expression [138]. These studies supported the notion that Nrf2 activation is critical for the protective mechanism of daphnetin against oxidative injury and inflammation induced by several insults. 3.7. Esculin. Esculin (7-hydroxy-6-[(2S,3R,4S,5S,6R)-3,4,5trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-2-one) is a coumarin derivative found in Aesculus hippocastanum L. (horse-chestnut). The anti-inflammatory [139] and antioxidant [140,141] activities of esculin have been wellacknowledged. By using the in vitro ABTS, ORAC, and DPPH assays, Zhang et al. have demonstrated the potent antiradical activity of esculin [141]. In a rat model of colon carcinogenesis, esculin mitigated oxidative stress, DNA damage, and tumorigenesis [140]. Additionally, esculin showed wide pharmacological activities against different diseases, such as cognitive impairment in experimental diabetic nephropathy where it exhibited a strong anti-inflammatory activity marked by the suppressed p38 MAPK and JNK [142]. In models of streptozotocin-induced renal damage in diabetic mice [143], cold-restrained stress and pylorus ligation-induced ulcer [144], ethanol-induced gastric lesion [145], and LPS/D-galactosamine-induced acute liver injury [146], esculin significantly ameliorated inflammation evidenced by the suppressed TNF-α, IL-1β, and MPO. Li et al. reported the protective role of esculin against LPS-induced macrophages and endotoxin shock in mice and NO production in vitro. Esculin inhibited the LPS-induced increase in 6 Oxidative Medicine and Cellular Longevity TNF-α and IL-6 and upregulated IL-10 via suppression of NF-κB [147]. Another study by Li et al. concluded that esculin markedly inhibited iNOS/NO levels, and NF-κB protein expression in gastric injury induced by alcohol [145]. Additionally, pretreatment with esculin suppressed TNF-α and IL-6 expression [145].
Pertaining to the impact of esculin on Nrf2 activation, Liu et al. have shown that esculin attenuated acute liver injury in mice induced by LPS/D-galactosamine and reduced pathological symptoms of acute hepatic injury via suppression of NF-κB expression as well as activation of Nrf2/HO-1 signaling [146]. Another study by Kim et al. reported that esculin activated Nrf2/ARE signaling in macrophages [148]. Additionally, esculin markedly inhibited neutrophilic lung inflammation which was not recapitulated in Nrf2 knockout mice, suggesting that the anti-inflammatory activity of esculin mainly acts via Nrf2 activation [148].
3.8. Esculetin. Esculetin (6,7-dihydroxychromen-2-one) is one of the main bioactive ingredients of Cortex Fraxini. Esculetin exhibits a potent antioxidant effect and showed a scavenging activity against DPPH radicals in a time-and concentration-dependent manner [149]. Esculetin has been widely used in antitussive aspects [150], and the study of Liang et al. has demonstrated its protective effect against oxidative stress-induced DNA damage [151]. In addition, esculetin possesses anti-inflammatory [152,153], antibacterial [154,155], and antitumor activities against different cancer cells in vitro [156][157][158] and in vivo [159,160]. It has also been reported to enhance the inhibitory effect of 5-fluorouracil on the proliferation of colorectal cancer [161]. The antiinflammatory activity of esculetin was effective in the inhibition of cartilage destruction in rheumatoid arthritis and osteoarthritis where it suppressed MMP-1 expression in cartilage and decreased NO and PGE2 levels in the synovium [162]. In the context of obesity, esculetin attenuated chronic inflammation by suppressing proinflammatory cytokine release during the interaction between adipocytes and macrophages [163]. In psoriatic mouse skin, esculetin attenuated the disease progression and dramatically decreased proinflammatory cytokines, including TNF-α, IL-6, IL-22, IL-23, IL-17A, and IFN-γ [152].
Several studies have supported the involvement of Nrf2 signaling in the pharmacological activities of esculetin. For instance, the study of Rubio et al. illustrated the different roles of NF-κB and Nrf2 in the antioxidant imbalance produced by esculetin on leukemia cells and concluded that esculetin resulted in a significant increase in the nuclear translocation of Nrf2 [164]. pathway [167]. Treatment of the pancreatic cancer cell lines with esculetin resulted in significant inhibition of cell proliferation, intracellular ROS, and protein levels of NF-κB [168]. Additionally, esculetin increased the Nrf2 and NQO1 gene expression as well as Nrf2 nuclear accumulation and induced mitochondrial-dependent apoptosis [168]. The same study has shown the binding ability of esculetin to directly bind Keap1 as evidenced by molecular docking and in vitro assays [168]. Subramaniam and Ellis reported that esculetin protects HepG2 cells against H 2 O 2 -induced injury via activation of the Nrf2/NQO1 pathway [169]. Pretreatment of the HepG2 cells with esculetin preserved cell integrity following exposure to H 2 O 2 and increased the nuclear accumulation of Nrf2 [169].
Several researchers have demonstrated the strong anti-inflammatory activity of UMB. The in vivo antiinflammatory activity of UMB has recently been reported by Wang et al. who showed its protective effect against acute lung injury induced by LPS [179]. Another study by Yin et al. reported that UMB inhibited inflammation in diabetic mice through suppressing NF-κB and TLR-4. In addition, UMB mitigated hepatic oxidative injury via activating the Nrf2mediated signal pathway [180]. Li et al. reported that UMB significantly attenuated ROS accumulation and cytotoxicity induced by methylglyoxal (MG) through the activation of Nrf2/ARE signaling [181]. Depletion of Nrf2 by siRNA markedly inhibited the protective effect of UMB against MG-induced alterations, suggesting the key role of Nrf2 in mediating UMB's activity [181]. The role of Nrf2 has been supported by the study of Sen et al. showing that UMB attenuated the progression of diabetic nephropathy via Nrf2 activation and inhibition of ROS production induced by HG [166]. Additionally, Mohamed et al. reported that UMB ameliorated CCl 4 -induced hepatotoxicity in rats through induction of the nuclear translocation of Nrf2, thereby ameliorating oxidative stress-related liver injury via enhancement of cellular antioxidant defenses [138]. Furthermore, UMB protected against renal injury induced by methotrexate (MTX) and attenuated oxidative injury via downregulation of Keap1 and upregulation of Nrf2. The results have also shown that UMB inhibited inflammatory responses via downregulation of both NF-κB and p38 MAPK in the kidney of MTX-intoxicated rats [174].
Previous work from our lab has demonstrated the antioxidant and anti-inflammatory activities of UMB in different animal models [30,34,44]. UMB administration mitigated cyclophosphamide-induced oxidative damage and inflammatory response through marked elevation of Nrf2, HO-1, and PPARγ expression. UMB attenuated lipid peroxidation, enhanced antioxidants, and suppressed serum proinflammatory mediators and hepatic iNOS and NF-κB expression. The results showed that coactivation of Nrf2 and PPARγ represents the main mechanism underlying the hepatoprotective effect of UMB [44]. In a rat model of CCl 4 -induced hepatic fibrosis, UMB mitigated inflammation, oxidative injury, and collagen deposition. In addition, UMB suppressed NF-κB p65 and TGF-β1/Smad3 and upregulated hepatic PPARγ [34]. Moreover, UMB attenuated lipid peroxidation, NO release, and cerebral inflammation and downregulated nNOS and soluble guanylate cyclase expression in the cerebrum of hyperammonemic rats [30].
The role of Nrf2 in mediating the pharmacologic effects of fraxetin has been demonstrated in few studies. In this context, the protective effect of fraxetin on oxidative stress induced by Plasmodium berghei infection in mice has been investigated by Singh et al. [193]. Postinfection treatment of the mice with fraxetin suppressed lipid peroxidation and boosted GSH and antioxidant enzymes. The authors have also reported a significant increase in the serum Nrf2antioxidant response element level [193]. The effect of fraxetin on HO-1 expression in HaCaT human keratinocytes has been investigated by Kundu et al. [194]. Fraxetin activated the Nrf2/HO-1 pathway in HaCaT cells, induced the nuclear translocation of Nrf2, and increased the ARE-reporter gene activity [194]. Thuong et al. reported that fraxetin inhibited vascular proliferation and atherosclerosis and upregulated HO-1 in vascular smooth muscle cells (VSMCs) [195]. Subcellular fractionation and reporter gene analysis using an ARE construct demonstrated that fraxetin upregulated Nrf2 and reporter activity and concluded that fraxetin has direct protective properties against LDL oxidation via Nrf2/ARE activation [195].
The dual antioxidant and anti-inflammatory activity of fraxin has been recently demonstrated in a mouse model of acute respiratory distress syndrome (ARDS) [200]. Fraxin inhibited the production of TNF-α, IL-1β, IL-6, ROS, and MDA; suppressed NF-κB, MAPK signaling, and MMP9; and increased SOD in the lung of mice with LPS-induced ARDS [200]. Niu et al. reported that fraxin exhibited hepatoprotective effects against CCl 4 -induced liver damage via mitigation of oxidative stress and inflammation [197]. Fraxin alleviated hepatic injury as indicated by the suppressed production of inflammatory mediators and enhancement of the antioxidant defense mechanisms. An in vitro study demonstrated that pretreatment of HepG2 with fraxin protected against the deleterious effects of CCl 4 . Fraxin inhibited CCl 4 -induced MAPK, NF-κB, and COX-2 protein expression [197]. In two different studies conducted by Li et al., the protective effect of fraxin against LPS-induced endotoxic shock [201] and acute lung injury in mice [202] has been investigated. In both studies, the ameliorative effect of fraxin was associated with decreased release of proinflammatory mediators, suppressed ROS generation and oxidative stress, and downregulated NF-κB and NLRP3 inflammasome signaling pathways. These findings highlighted the potent suppressive effect of fraxin on both inflammatory and oxidative responses [201,202]. In an animal model of I/R-induced kidney injury, fraxin exhibited an ameliorative effect mediated through suppressing oxidative DNA damage and NF-κB [203]. Furthermore, Chang et al. have pointed to the role of Nrf2 in mediating the hepatoprotective efficacy of fraxin [198]. In their study, fraxin markedly inhibited the t-BHPinduced cytotoxicity and ROS generation in HepG2 through Nrf2 pathway-dependent HO-1 expression. In vivo studies showed that fraxin has potent hepatoprotective effects against CCl 4 -induced hepatotoxicity in rats via direct antioxidant activity and the Nrf2/ARE pathway [198].
Studies showing the modulatory effect of wedelolactone on Nrf2 signaling are very few. Lin et al. reported that wedelolactone inhibited t-BHP-induced damage in PC12 cells and D-galactose-induced neuronal cell loss in mice through improvement of the antioxidant defense capacity via Nrf2/ARE pathway activation [219]. On the other hand, Ding et al. demonstrated that wedelolactone protected human bronchial epithelial cells against cigarette smoke extractinduced oxidative stress and inflammation responses through Nrf2 inhibition [220].
3.15. Osthole. Osthole (7-methoxy-8-(3-methylbut-2-enyl)chromen-2-one) is a coumarin found in a high content in the mature fruit of Cnidium monnieri which is commonly applied in the clinical practice of Chinese medicine. Osthole is also widely distributed in other medicinal plants of the genera Citrus, Clausena Angelica, and Archangelica. Fructus Cnidii improved male function and reinforced the immune system mainly due to its rich content of osthole [227,228].
Several reports of underlying molecular mechanisms reported that osthole displays a strong anti-inflammatory activity. Fan et al. reported that osthole effectively and safely protected against ulcerative colitis (UC) via marked inhibition of TNF-α expression in the colon and markedly reduced MPO activity via suppression of NF-κB p65 and p-IκBα [234]. In an in vitro study, osthole inhibited the production of TNF-α, NO, PGE2, and IL-6 in LPS-induced macrophages [234]. Osthole has a potent selective inhibitory effect on 5lipoxygenase and COX-1 [236,237]. It suppressed the immune response of LPS-stimulated macrophages by abolishing ROS generation, iNOS, MAPK, and COX-2 [238,239]. Osthole also suppressed IL-4-and TNF-α-induced eotaxin expression in bronchial epithelial cells [240] and protected against carrageenan-induced hind paw edema in rats via suppression of PG and NO production [241]. Osthole protects against lumbar disc herniation-induced sciatica and relieved mechanical allodynia through decreasing the COX-2 and iNOS expression in the dorsal root ganglion in rats [242].
The modulatory effect of osthole on Nrf2 signaling has been well-acknowledged by different researchers. A recent study conducted by Chu et al. reported that the protective effect of osthole against glutamate-induced Alzheimer's disease in mice was mediated via Nrf2 activation and its downstream antioxidant proteins SOD-1 and HO-1 [243]. Osthole protected against Ang II-induced apoptosis of rat aortic endothelial cells through suppression of NF-κB and activation of Nrf2 and its downstream antioxidant genes and effectively inhibited Keap1, denoting its potential therapeutic effect against vascular injury [244]. In another study, osthole protected against LPS-induced inflammation in BV2 cells via NF-κB suppression and upregulation of the Nrf2/HO-1 pathway dose dependently [245]. Additionally, osthole exerted neuroprotective effects against global cerebral I/R injury by reducing oxidative stress via the upregulation of the Nrf2/HO-1 signaling pathway [246]. The renoprotective effect of osthole against accelerated focal segmental glomerulosclerosis was mediated via Nrf2 activation and subsequently downregulation of the NF-κB-mediated COX-2 expression [247]. Osthole has also shown renoprotective effects mediated through inhibition of ROS generation and NF-κB/NLRP3 signaling and increased Nrf2 nuclear translocation [248]. Osthole has protective effects on LPS-induced acute lung injury by upregulating the Nrf-2/Trx-1 pathway, whereas Nrf2 siRNA blocked its beneficial effects [249].
The effects of coumarins on Nrf2 signaling in in vivo and in vitro studies are summarized in Tables 1 and 2, respectively.

In Silico Evidence for Binding of Coumarins to Keap1 Protein
Keap1 plays the key step in the ubiquitination and degradation of Nrf2. In this review, we aimed to provide in silico evi-dence that coumarins bind Keap1 and hence could be employed as promising Nrf2 activators. AutoDock Vina 1.5.6 was used to perform molecular docking of coumarin derivatives and Keap1 protein. The complex structure of Keap1 with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1Hisoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)) with PDB ID: 4l7b was used as a model for the docking study ( Figure 3). The binding pocket of Keap1 was used to identify the binding conformation of the different coumarin derivatives ( Figure 4). The average of the lowest energy of docking was used to show the binding affinity for each coumarin derivative with Keap1. The best-scored conformation has been chosen and visually analyzed using the PyMOL 1.7.6 software. Briefly, 9 different orientations were generated, and the first pose with the lowest docking energy has been used. Each coumarin has a different conformation; however, all the conformations were allocated in the vicinity of the active site. The mean docking energy of all 9 generated models was calculated. There are very limited biophysical studies that include the experimental binding data of all listed coumarin derivatives and Keap1. Therefore, this review sheds light on the promising compounds which should be studied in the future. To perform an experimental study, the Keap1 protein needs to be expressed and purified, and the binding affinities with different coumarin derivatives could be estimated using isothermal titration calorimetry (ITC) and/or surface plasmon resonance (SPR). Based on the binding affinity, crystallization trials could be conducted to get an accurate binding mode between Keap1 and coumarins. This review highlights the promising target for biophysical studies.
IMP: the carbonyl group of a coumarin moiety forms a hydrogen bond and n ⟶ π * interaction with the amino group and carbonyl group of NH 2 of the side chain of N414, respectively. Also, the oxygen atom of furan and pyran rings forms two hydrogen bonds with the -OH group of the side chain of S602 and S363, respectively.
Visnagin: the carbonyl group and the oxygen of the methoxy group of the coumarin moiety form two hydrogen bonds with the -OH group of the side chain of S602, while the oxygen of the furan ring forms a hydrogen bond with the amino group of the side chain of N387 at the dimeric interface of the Keap1 protein. [250] Isoimperatorin Aflatoxin B1-inducible cytotoxicity in H4IIE Isoimperatorin activated Nrf2/ARE and GST-α and suppressed CYP1A expression. [256] Binding pocket

Oxidative Medicine and Cellular Longevity
Urolithin B: the carbonyl and hydroxy groups of the coumarin moiety exhibited two hydrogen bonds with S602 and S363, respectively. These two hydrogen bonds have a remarkably short length approximately 2.9 Å and 2.7 Å which could enhance the interaction of urolithin B and binding pocket of Keap1.
Urolithin A: this forms five hydrogen bonds at the dimeric interface of the Keap1 protein. Both hydroxyl groups form hydrogen bonds with S363, P384, and S383. Also, the carbonyl groups form two hydrogen bonds with the -OH group of the side chain of Y334 and the amino group of the side chain of N387.
Daphnetin: the carbonyl oxygen forms a hydrogen bond with S602, and the two hydroxy groups form two hydrogen bonds with S363.
Esculin: this coumarin is rich in hydroxyl groups which serve as hydrogen bond donors to the residues in the active site of Keap1. It forms six hydrogen bonds with the carbonyl group of C368, A366, V418, V604, and V606. These hydroxyl groups of the oxan ring support the perfect fitting of esculin deep in the binding pocket of Keap1. In addition, the hydroxyl group of the coumarin moiety forms two hydrogen bonds with V463 and A510. The carbonyl group of the lactone ring forms a hydrogen bond with the amino group of the side chain of R415. Esculin makes hydrophobic interactions with the nonpolar amino acids in V418, V604, V606, V463, and A510.
UMB: this forms two hydrogen bonds with S363 and N387 at the dimeric interface of Keap1.
Fraxetin: the dihydroxy groups form bonds with the side chain of N382 and S363, and the oxygen of the methoxy group forms a hydrogen bond with S363. The carbonyl group and oxygen of the pyran ring form two extra hydrogen bonds with N387. These five hydrogen bonds are located at the dimeric interface of Keap1.
Fraxin: the hydroxyl groups of oxan rings and oxygen of the ether bond between the oxan ring and the coumarin nucleus form three hydrogen bonds with the side chain of R415, S555, and S602. Also, the hydroxyl and carbonyl groups of this coumarin form two hydrogen bonds with S602 and S363, respectively. The coumarin nucleus exhibits a different conformation in fraxin as compared to esculin which could prevent the perfect fit of fraxin in the binding pocket of Keap1. This explanation is supported by the lower energy of the docking of esculin than fraxin.
Esculetin: this forms six hydrogen bonds with S602, N382, N387, and Y334. Also, the benzene ring of the coumarin nucleus makes hydrophobic interaction with the aromatic ring of Y334.
Anomalin: this forms two hydrogen bonds with R415 and S602.
Wedelolactone: this fits properly in the active site of Keap1 due to its unique conformation. The hydroxyl group of the coumarin moiety forms two hydrogen bonds with the carbonyl groups of L365 and V604. The carbonyl group of coumarin exhibits n ⟶ π * interaction with the carbonyl  14 Oxidative Medicine and Cellular Longevity group of A510. The two-hydroxyl group of the benzofuran ring forms two hydrogen bonds with the side chains of R415. Wedelolactone has hydrophobic interactions with L365, A366, V604, and A510. Glycycoumarin: the oxygens of the methoxy and carbonyl groups of coumarin form two hydrogen bonds with R415 and S363. Another two hydrogen bonds are formed between the hydroxy group of the dihydroxyphenyl group and the side chain of N382 and Y334. The phenyl group of glycycoumarin has a hydrophobic interaction with the aromatic ring of the side chain of Y334.
Scopoletin: this forms six hydrogen bonds with the side chains of S363, N414, N382, and Y334 at the dimeric interface of Keap1. Also, there is n ⟶ π * interaction between the carbonyl group of coumarin and the carbonyl group of R336.
Osthole: this forms three hydrogen bonds with the side chains of N387 and S602 at the dimeric interface of Keap1.
Hydrangenol: this forms three hydrogen bonds with I559 and the side chain of R415. It has a hydrophobic interaction with A556, V604, I559, and L577.
Isoimperatorin: this forms two hydrogen bonds with the side chains of S602 and N387 at the dimeric interface of Keap1.

Conclusions
The Nrf2 signaling pathway plays a vital role in the protection against cell injury induced by oxidative stress and elec-trophiles. Excessive production of ROS and oxidative injury are associated with the pathogenesis of several diseases and disorders. There is crosstalk between the Nrf2/ARE signaling and other signaling pathways, including NF-κB, and this represents the main underlying mechanism by which Nrf2 exerts its anti-inflammatory activities. Given the dual role of Nrf2 activation in the prevention of oxidative stress and inflammation, pharmacological activation of this signaling pathway could represent a powerful strategy for treating diseases associated with excessive release of ROS and proinflammatory mediators. Coumarins represent a vast group of natural compounds with beneficial pharmacological properties. Several studies have demonstrated the role of Nrf2 activation in mediating the therapeutic effects of some coumarins both in vitro and in vivo. We employed molecular docking simulations to investigate the binding of coumarins with Keap1 and to verify whether their therapeutic effects are related to antioxidant properties mediated via Nrf2 modulation. IMP, visnagin, urolithin B, urolithin A, scopoletin, esculin, esculetin, UMB, fraxetin, fraxin, daphnetin, anomalin, wedelolactone, glycycoumarin, osthole, hydrangenol, and isoimperatorin showed binding affinities toward Keap1 through forming hydrogen bonds with the side chains of the polar, positively, and negatively charged amino acids. IMP, urolithin B, urolithin A, esculin, fraxin, wedelolactone, glycycoumarin, and hydrangenol exhibited strong binding with Keap1 with affinities quite close to a standard Keap1 inhibitor. Thus, activation of the Nrf2 signaling pathway can explain some of the pharmacological and biological effects of coumarins. Coumarins therefore represent promising leads in the development of effective Keap1 inhibitors/Nrf2 activators and a good resource for discovering drug candidates for the treatment/prevention of various diseases.

Conflicts of Interest
The authors report no conflict of interests.